Lifetime technology: time to get excited!

fluorescence lifetime

Fluorescence is the emission of light from a substance upon excitation by higher energy radiation. Fluorescence lifetime is the average time observed for a population of molecules to return from the excited to the ground state. A variety of processes can accelerate the return to the ground state by offering additional pathways into which the excited-state energy can flow, yielding shorter observed fluorescence lifetimes. These processes are called dynamic quenching, and they form the basis for the FLEXYTE® assays that utilize fluorescence lifetime as their readout signal.

Fig 1A Fig 1B

Fig 1: A. Schematic detailing the emission of light following return of molecules from the excited to ground state. A quencher (Q) can modulate this process by providing an additional decay pathway. B. Typical fluorescence decay curves for control and quenched samples (semi-log plot). Following excitation, the recorded fluorescence decays exponentially. The fluorescence lifetime can be calculated as the inverse of the slope of the decay curve.

fluorescence lifetime characteristics

Interferences encountered in light-based biochemical assays are often due to the uncertainty in the number of observable molecules, stemming from variations in sample volume or concentration, or through masking by sample constituents. In contrast, fluorescence lifetime is independent of the amount of observable sample (Fig 2). This property offers a much increased assay robustness compared to traditional fluorescence readouts.

Fig 2A Fig 2B

Fig 2: A. Fluorescence lifetime is independent of the number of molecules in the sample. B. Such independence confers significant robustness to lifetime-based technologies in contrast to intensity-dependent readouts.

what integrated lifetime screening platform is available?

The platform has been developed in conjunction with major pharma partners and is designed for easy adoption into existing drug discovery workflows. A broad range of therapeutic targets can be addressed for lead identification and optimization activities.

Fig 3: The lifetime technology platform

what is different about lifetime technology as a screening method?

When selecting a fluorescence-based assay technology, two important considerations are the need for a large assay window and good discrimination from assay interferences. Lifetime technology utilises the FLEX17 fluorophore, a 9-aminoacridine-based reporter excitable at 405 nm (Fig 4). FLEX17 has a fluorescence lifetime of 17 ns when unquenched. This is exceptionally long when compared to traditional fluorophores (FITC, 4 ns; Cy5, 1 ns; AlexaFluor 647, 1 ns), offering ample scope for achieving wide assay windows. Additionally, this long lifetime is well separated from the majority of fluorescent assay interferences. Thus, compared to other fluorescence-based approaches, lifetime is largely immune to compound-related assay interferences and is therefore able to offer a better detection of false negatives and rejection of false positives.

Fig 4: Structure of the FLEX17 long lifetime fluorophore employed in FLEXYTE assays.

assay principles

FLEXYTE assay configurations are centred around the modulation of FLEX17’s lifetime, by incorporating a suitable dynamic quencher in close proximity to the label. In the most elegant case, the quencher is an aromatic residue (tyrosine or tryptophan) located on the same peptidic substrate (Fig 5). The presence of the quencher significantly reduces the lifetime of FLEX17 providing a stable and large assay window.

In a more complex case, such as monitoring protein-protein interactions, FLEXYTE lifetime assays comprise a FLEX17 labelled protein and a ligand coupled to a Delta™ lifetime modulator. Upon ligand binding, the Delta lifetime modulator is brought into close proximity to the FLEX17 fluorophore, reducing its lifetime (Fig 6). Compounds that disrupt this protein-protein interaction are detected by an increase in fluorescence lifetime.

Fig 5 Fig 6

Fig 5: The FLEXYTE lifetime tyrosine protein kinase assay uses FLEX17-labelled peptide substrates. The presence of a tyrosine residue in the substrate sequence directly modulates the FLEX17 lifetime. Phosphorylation of this tyrosine by a corresponding kinase alleviates the quenching, leading to an increase in lifetime which correlates directly with the level of phosphorylation of the substrate.

Fig 6: FLEXYTE protein-protein interaction lifetime assay principle.

FLEXYTE lifetime reagents and assays

FLEXYTE lifetime reagents enable antibody-free and cost effective screening solution based on fluorescence lifetime reporting. They assist you by improving the precision of your compound screening while still using your existing HTS workflows. The intrinsic robustness of FLEXYTE lifetime reagents reduces compound interference and selects fewer false leads, with a reliability normally associated with biophysical methods. This increases the efficiency of compound screening while controlling costs, making it easy to increase your drug discovery productivity. FLEXYTE lifetime reagents are available as fully configured assays for a broad range of different target classes (Fig 7).